Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.     

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation.     

Analysis of type II collagen RNA localization in chick wing buds by in situ hybridization

Type II collagen is a major component of cartilage extracellular matrix. Differentiation of mesenchyme into cartilage involves the cessation of type I collagen synthesis and the onset of type II collagen synthesis. Solution hybridization of mRNA isolated from chick limb buds with a cDNA probe to type II collagen mRNA showed the presence of small amounts of type II collagen message in mesenchymal chick limbs. We have examined the localization of type II collagen mRNA in mesenchymal chick wing buds by in situ hybridization using single stranded RNA probes. Our results show a small but detectable amount of type II collagen RNA distributed uniformly in early limbs until the first precartilage condensations form at stage 22. This is interesting because it is known that mesenchyme isolated from chick wing buds has the capacity to undergo chondrogenesis in culture, even if taken from nonchondrogenic areas of the limb. At stage 23, type II collagen mRNA is found at significantly increased levels in the cells of the precartilage condensation when compared to the other limb cells. As chondrogenesis proceeds, the amount of type II collagen RNA increases even more in cells of the cartilage elements. The signal in the peripheral tissue is indistinguishable from background. These results show that type II collagen message exists at low levels in cells throughout the mesenchymal chick wing bud, until the formation of the condensation results in an elevation of type II mRNA in the prechondrogenic cells found in the core of the limb.     

Effect of 4-methylumbelliferyl-β-d-xyloside on collagen synthesis in chick limb bud mesenchymal cell cultures

Previous studies showed that cultures of chick limb bud mesenchymal cells plated at high density, to maximize chondrogenic expression, had a much reduced extracellular matrix around chondrocytes when exposed to 4-methyl-, umbelliferyl-β-d-xyloside. The majority of newly synthesized chondroitin sulfate chains were found in the culture medium presumably bound to the xyloside as opposed to their normal deposition on the core protein of proteoglycan. The question remained open as to whether the development of an abnormal matrix affected the synthesis of extracellular deposition of other cartilage-specific macromolecules. We have analyzed, both morphologically and biochemically, the synthesis and deposition of Type I and Type II collagen by β-d-xyloside-treated cultures of limb mesenchymal cells. While the rate of collagen synthesis per plate and its extracellular accumulation after 8 days in culture were reduced to some extent, the ratios of Type II to Type I collagen and the morphological distribution of these macromolecules were not affected by exposure to β-d-xyloside. We conclude that the expression of the cartilage-specific Type II collagen during chondrogenic differentiation is, although reduced, qualitatively not dependent on the amount of extracellular chondroitin sulfate chains attached to matrix-associated proteoglycan core protein. However, prolonged exposure of limb bud cells to xylosides leads to the formation of a chondroitin sulfate- and collagen-deficient matrix which, in turn, reduces the capacity of limb bud cells to synthesize Types I and II collagen.     

Two distinct classes of prechondrogenic cell types in the embryonic limb bud

Differences are demonstrated in the chondrogenic potential of cells derived from the distal and proximal halves of chick wing buds from as early as stage 23, prior to the appearance of overt cartilage differentiation. In high cell density cultures, cells obtained from the distal portions of stage 23 or 24 limb buds are spontaneously chondrogenic in micromass cultures. Cells obtained from the proximal portions, however, become blocked in their differentiation as protodifferentiated cartilage cels, since these cells in micromass cultures make detectable type II collagen, but fail to synthesize significant levels of cartilage proteoglycan or to accumulate an extracellular matrix that will stain for sulfated glycosaminoglycans. Such cultures of proximal limb bud cells can be stimulated to form alcian blue staining nodules by the addition of 1 mM dbcAMP or 50 micrograms/ml ascorbate, or by mixing proximal cells with small numbers of distal cells (1 distal cell to 10 proximal cells). These results demonstrate the existence of two distinct stages among prechondrogenic mesenchyme cells. The earlier stage appears to be able to provide a chondrogenic stimulus to proximal cells.     

Cartilage proteoglycan core protein gene expression during limb cartilage differentiation

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant two- to fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to cAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes.     

A radioimmune study of the effect of bromodeoxyuridine on the synthesis of proteoglycan by differentiating limb bud cultures.

A radioimmune assay has been developed for the quantitative determination and the qualitative identification of core protein of cartilage chondroitin sulfate proteoglycan. Utilizing this method it has been shown that during differentiation of chick limb bud mesenchyme to cartilage, there is a marked augmentation of synthesis of core protein. Treatment with 5-bromo-2′-deoxyuridine results in an irreversible inhibition of synthesis of cartilage-specific chondroitin sulfate proteoglycan.     

Immunological characterization of the major chick cartilage proteoglycan and its intracellular localization in cultured chondroblasts: a comparison with Type II procollagen

Polyclonal antibodies were raised in a rabbit against the major proteoglycan of chick sternal cartilage. A total of six antisera was obtained, three after the first booster injection (A1, A2, and A3) and three after the second booster injection (A4, A5, and A6). The A1 antiserum, which was characterized in most detail, immunoprecipitated native as well as chondroitinase ABC-digested or chondroitinase ABC/keratanase-digested cartilage proteoglycan synthesized by cultured chick chondroblasts, but failed to immunoprecipitate the major proteoglycan synthesized by chick skin fibroblasts. This antiserum was also able to immunoprecipitate the cartilage proteoglycan core protein newly synthesized by cultured chondroblasts, but no other major cell protein. However, the late bleed antisera obtained from the same rabbit after a second booster injection reacted with a new chondroblast- specific polypeptide(s) of approximately 60,000 mol wt in addition to the cartilage proteoglycan. By immunofluorescence procedures, the A1 antiserum stained the extracellular proteoglycan matrix of cultured chondroblasts but not that of skin fibroblasts. Following enzymatic removal of the extracellular matrix and cell membrane permeabilization, this antiserum stained primarily a large, juxtanuclear structure. Additional radioautographic evidence suggests that this structure represents the Golgi complex. Similar immunofluorescent staining with antibodies to the cartilage-characteristic Type II collagen revealed that type II procollagen was localized in numerous cytoplasmic, vacuole- like structures which were scattered throughout most of the chondroblast cytoplasm but were notably scanty in the Golgi complex area. In conclusion, our data suggest the transit of the major cartilage proteoglycan through the Golgi complex of cultured chondroblasts and possible differences in the intracellular distribution of newly synthesized cartilage proteoglycan and Type II procollagen.     

Temporal and spatial expression of genes for cartilage extracellular matrix proteins during avian mandibular arch development

We have examined the temporal expression of genes for extracellular matrix proteins (type I collagen, type II collagen, and the cartilage specific proteoglycan core protein) during the development of the avian mandibular arch. We detected low levels of type II collagen mRNA in the mandibular arch as early as stage 15. Type II collagen mRNA remained low but increased slightly as development progressed from stage 15 to stage 25. More dramatic increases occurred after stage 25 coincident with overt chondrogenesis. In contrast, mRNA for the core protein of cartilage specific proteoglycan was not detected prior to the onset of chondrogenesis, appeared at stage 25, and increased thereafter. Type I collagen mRNA was also present as early as stage 15 and dramatically increased after stage 28/29, coincident with initiation of osteogenesis. Using in situ hybridization, we found that type II collagen mRNA became detectable in the center of the mandible around stage 24/25 coincident with the initiation of chondrogenesis. At later stages (26-32) type II collagen mRNA was localized in the cartilaginous rudiment. The pattern of hybridization observed with the proteoglycan core protein probe at later stages of development was essentially identical to that observed with the type II collagen probe. In contrast, the probe for the alpha 1 (I) collagen mRNA was localized over the perichondrium, over differentiated bone, and in areas within the mandibular arch where bone formation had been initiated.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.     

BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture

Human bone marrow-derived mesenchymal stem cells (MSCs) have been shown to differentiate into distinct mesenchymal tissues including bone and cartilage. The capacity of MSCs to replicate undifferentiated and to mature into cartilaginous tissues suggests these cells as an attractive cell source for cartilage tissue engineering. Here we show that the stimulation of human bone marrow-derived MSCs with recombinant bone morphogenetic protein-2 (BMP2) results in chondrogenic lineage development under serum-free conditions. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining of cartilage-specific type II collagen revealed the deposition of typical cartilage extracellular matrix components. Semi-quantitative real-time gene expression analysis of characteristic chondrocytic matrix genes, such as cartilage link protein, cartilage oligomeric matrix protein, aggrecan, and types I, II, and IX collagen, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with BMP2 and transforming growth factor-beta3 (TGFbeta3). Histologic staining of mineralized extracellular matrix with von Kossa, immunostaining of type X collagen (typical for hypertrophic chondrocytes), and gene expression analysis of osteocalcin and adipocyte-specific fatty acid binding protein (aP2) further documented that BMP2 induced chondrogenic lineage development and not osteogenesis and/or adipogenesis in human MSCs. These results suggest BMP2 as a promising candidate for tissue engineering approaches regenerating articular cartilage on the basis of mesenchymal progenitors from bone marrow.     

Biosynthesis of type IX collagen during chick limb development

We analyzed the collagens synthesized by developing chick limbs (stages 22 to 34). Type IX collagen synthesis started at stage 26, concurrently with the chondrogenic differentiation of limb mesenchyme, and gradually increased during subsequent stages. By stage 34, the central cartilaginous region of the limbs substantially synthesized type IX collagen, in addition to cartilage-specific type II collagen, while the outer non-cartilaginous region of the limbs synthesized predominantly type I collagen. The present study indicates that type IX collagen is cartilage-specific and can be used as a marker for the chondrogenic phenotype.     

An immunofluorescence study of chondrogenesis in murine mandibular ectomesenchyme

The temporal and spatial distribution of type I collagen, type II collagen, cartilage-specific proteoglycan (CSPG) and fibronectin in mouse mandible is described. CD-1 mouse embryos of 12-, 15-, and 18-day gestation were used, and matrix molecules were localized using indirect immunofluorescence. On day 12, accumulation of type II collagen, CSPG, and fibronectin within regions of condensed mesenchyme was noted. On day 15, intense staining for type II collagen and CSPG occurred. Fibronectin was less brilliant with its greatest concentration near the perichondrium. On day 18, the cartilage matrix was undergoing osseous replacement concurrent with loss of type II collagen and CSPG. Type I collagen was seen in the perichondrium, membranous bone and sub-basement membrane region in specimens of all ages. Synthesis and expression of extracellular matrix molecules reflect patterns of differentiation in mandibular mesenchyme.     

Correction of abnormal matrix formed by cmd/cmd chondrocytes in culture by exogenously added cartilage proteoglycan

The cartilage matrix deficiency (cmd/cmd) mouse fails to synthesize the core protein of cartilage-characteristic proteoglycan (cartilage PG). Chondrocytes from the cmd/cmd cartilage cultured in vitro produced nodules with greatly reduced extracellular matrix. Immunofluorescence staining revealed that the nodules of mutant cells differed from the normal in lacking cartilage PG and in uneven and reduced deposition of type II collagen. Exogenously added cartilage PG prepared from either normal mouse cartilage or Swarm rat chondrosarcoma to the culture medium was incorporated exclusively into the extracellular matrices of the nodules, with a concurrent correction of the abnormal distribution pattern of type II collagen. The incorporation of cartilage PG into the matrix was disturbed by hyaluronic acid or decasaccharide derived therefrom, suggesting that the incorporation process involves the interaction of added proteoglycan with hyaluronic acid. Both the hyaluronic acid-binding region and the protein-enriched core molecule prepared from rat chondrosarcoma cartilage PG could also be incorporated but, unlike the intact cartilage PG, they were distributed equally in the surrounding zones where fibroblast-like cells predominate. The results indicate that the intact form of cartilage PG is required for specific incorporation into the chondrocyte nodules, and further suggest that cartilage PG plays a regulatory role in the assembly of the matrix macromolecules.     

Nonuniform distribution of fibronectin during avian limb development

Using indirect immunofluorescence we have examined the distribution of the cell surface and extracellular matrix glycoprotein fibronectin at the epithelial-mesenchymal interface and in the mesenchyme of developing chick and duck wing buds. At all stages examined, in both species, staining for fibronectin is greatly enhanced in the basement membrane subjacent to the apical ectodermal ridge (AER), a site of inductive tissue interaction, relative to the epithelial basement membranes in the noninductive dorsal and ventral limb epithelial-mesenchymal interfaces. In stage 23, 25, and 28 chick limb buds, staining for fibronectin is uniform in the least mature distal mesenchyme, retained between more proximal cells undergoing precartilage condensation and lost in those regions undergoing myogenesis, and persistent in all but the most mature cartilage present at the latest stage examined. These results are consistent with a role for fibronectin in AER-induced limb outgrowth, and with a transient role for the glycoprotein in the formation of the skeletal pattern of the limb.